The binding of myosin subfragment-one (S-1) to the F-actin-troponin-tropomyosin complex (regulated F-actin) was examined in the presence of ADP at micron equals 0.22M, 22 degrees C, S-1 ADP binds with positive cooperativity to regulated F-actin, both in the presence and absence of calcium, while it binds independently to unregulated actin. With and without Ca2 ions at very low levels of occupancy of the regulated actin by S-1 ADP, S-1 ADP binds about 3-fold more strongly to the regulated actin than it does to unregulated actin. The major difference between the results obtained in the presence and absence of CA2 ions with regulated actin is that in the absence of Ca2 ions, the binding of S-1 ADP remains weak until a higher free S-1 ADP concentration is reached and the transition to strong binding is much more cooperative. These results are consistent with a cooperative binding model where the regulated actin filament can exist in two forms, a weak binding and a strong binding form with Ca2 ions and S-1 ADP as allosteric effectors shifting the equilibrium between the two forms. These studies also show that troponin-tropomyosin has the ability to greatly weaken the binding of S-1 ADP to actin, both in the presence and absence of Ca2 ions, although as shown in the accompanying annual report (00417-01) the troponin-tropomyosin has almost no effect on the binding of S-1 ATP to actin.